Development of biochemical and molecular-biological methods allowing relatively easy production of large amounts of recombinant proteins in high yields brought the need for their easy and particularly versatile isolation and visualization. Subsequent development of affinity (purification) tags allowed isolation, purification and visualization of various proteins always with the same technology, which meant a significant improvement in methodology of recombinant protein production and acceleration in their isolation and purification.
Polyhistidine sequence (also “tag”, abbreviated 6×His-tag), containing usually 6 histidines and known under the commercial name His-tag, is probably the most widely used and best known tag. The 6×His-tag sequence is relatively specifically bound with a complex containing chelating compound with a divalent cation of nickel or cobalt. This chelating compound may be e.g. nitrilotriacetic acid (NTA), which is the most commonly used for purification of proteins tagged with a polyhistidine sequence. Affinity can be further enhanced by using compounds containing more molecules of nitrilotriacetic acid, e.g. triNTA.
For the detection of the proteins with a polyhistidine sequence on Western blot, it is necessary to use antibody recognizing this sequence. The sensitivity of many commercially available antibodies against polyhistidine sequence is not sufficient for the quantification of small amounts of proteins labeled with polyhistidine sequences. The bond between an antibody and a polyhistidine sequence may generally not be strong enough, and thus dissociation of the complex may occur. This is a particular problem in methods requiring a very strong bond to immobilize proteins labeled with polyhistidine sequences, such as ELISA or surface plasmon resonance (SPR).
Glutathione-S-transferase (GST) is a glutathione binding enzyme and is involved in detoxification processes in the organism. GST can be expressed in fusion with the protein (GST-tag); this fusion protein can then be bound using a resin with glutathione. In addition to His-tag, GST-tag is another widely used affinity tag for protein purification.
Antibodies are large molecules of glycosylated proteins containing disulfide bonds, and their production is thus bound to a eukaryotic expression system, which allows to perform said post-translational modifications. For this reason, production of antibodies is relatively expensive.
Antibody molecules are also quite susceptible to degradation: as proteins they must generally be stored at low temperatures, and if necessary, frozen in aliquots. Their repeated thawing often leads to loss of their ability to bind a given antigen.
Polymers prepared by homopolymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) are biocompatible, nonimmunogenic and water-soluble. Thanks to these features, HPMA copolymers are used as carriers for drugs and imaging compounds, used particularly as anticancer drugs [1-2]. HPMA copolymers are multivalent macromolecules that can be linked with various low molecular compounds, e.g. fluorescent probes, radionuclides or drugs. Besides these low-molecular substances, HPMA copolymers can be modified with (glyco)proteins, oligonucleotides and polynucleotides. Multivalence of HPMA copolymers allows to connect both just one type of molecules, and combinations of various types with different functions [3-5].
The invention describes macromolecular conjugates of polymers capable of visualization, immobilization and separation of proteins labeled with purification tags.